南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (12): 1726-1731.doi: 10.12122/j.issn.1673-4254.2020.12.05

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Fractalkine 通过激活 Wnt/β-catenin 信号通路抑制脂多糖诱导的巨噬细胞M1型极化

巩奇明，姜 艳，卢俊玲，尤燕舞   

  • 出版日期:2020-12-20 发布日期:2020-12-28

Fractalkine inhibits lipopolysaccharide-induced M1 polarization of macrophages by activating Wnt/β-catenin signaling pathway

  • Online:2020-12-20 Published:2020-12-28

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摘要： 目的 研究Fractalkine（CX3CL1，FKN）对脂多糖（LPS）诱导的RAW264.7巨噬细胞免疫应答的作用机制。方法 体外培养RAW264.7细胞，使用慢病毒技术构建过表达FKN的稳定细胞珠。细胞分为8组：（1）空白对照组；（2）LPS组，细胞给予脂多糖（1 μg/mL，12 h）；（3）ICG-001组，细胞给与Wnt/β-catenin信号通路抑制剂ICG-001（10 μmol/mL，48 h）；（4）过表达FKN组；（5）ICG-001+LPS组，细胞给与ICG-001（10 μmol/mL预处理36 h）后，加入脂多糖（1 μg/mL，12 h）；（6）过表达FKN+LPS组，过表达
FKN细胞给与脂多糖（1 μg/mL，12 h）；（7）过表达FKN+ICG-001组，过表达FKN细胞给予ICG-001（10 μmol/mL，48 h）；（8）过表达 FKN+ICG-001+LPS 组，过表达 FKN 细胞给与 ICG-001（10 μmol/mL 预处理 36 h）后，加入脂多糖（1 μg/mL，12 h）；应用
CCK-8细胞增殖实验检测ICG-001作用于巨噬细胞中的安全浓度；应用酶联免疫吸附（ELISA）实验检测巨噬细胞上清液中M1型极化因子TNF-α和IL-6的含量；应用蛋白质免疫印记（WB）实验检测巨噬细胞中FKN、Wnt/β-catenin通路关键因子Wnt-4和β-catenin、M1型极化因子iNOS、TNF-α和IL-6的蛋白表达水平；应用免疫荧光（IF）实验检测巨噬细胞中M1型极化因子IL-6蛋白的定位。结果 过表达FKN的RAW264.7细胞中FKN的蛋白水平较空白对照组明显升高（P<0.01）。CCK-8实验显示ICG-001作用于RAW264.7细胞48 h的IC50为10 μmol/mL。与LPS组相比，ICG-001+LPS组上清液中TNF-α和IL-6的分泌量以及细胞内TNF-α、IL-6和iNOS蛋白含量均升高（P<0.05），而细胞内FKN、Wnt-4和β-catenin蛋白含量均显著降低（P<0.01）；EXFKN+LPS组上清液中TNF-α和IL-6的分泌量以及细胞内TNF-α、IL-6和iNOS蛋白含量均显著降低（P<0.01），而细胞内FKN、Wnt-4和β-catenin蛋白含量均显著升高（P<0.01）。与LPS组相比，ICG-001+LPS组中IL-6在细胞质中的定位增强，而EXFKN+LPS组中IL-6在细胞质中的定位受到抑制。结论 过表达FKN通过激活Wnt/β-catenin信号通路抑制脂多糖诱导的巨噬细胞M1型极化。

关键词: 巨噬细胞；极化；Fractalkine；Wnt/β-catenin信号通路

Abstract: Objective To explore the mechanism by which fractalkine (CX3CL1; FKN) inhibits lipopolysaccharide (LPS)-induced immunological response in RAW264.7 cells. Methods A RAW264.7 cell model overexpressing FKN was established by transfection with the lentiviral vector CX3CL1. The effects of LPS, ICG-001 (a Wnt/β-catenin signaling pathway inhibitor), either alone or in combination, on M1 polarization of na?ve and FKN-overexpressing RAW264.7 cells were evaluated by detecting of intereukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) using ELISA. The protein expressions of the inflammatory factors (iNOS, TNF-α, and IL-6), FKN, Wnt-4, and β-catenin were detected by Western blotting. The subcellular localization of IL-6 in the cells was detected by immunofluorescence assay. Results The RAW264.7 cell model of FKN overexpression was successfully established. In na?ve RAW264.7 cells, treatment with both ICG-001 and LPS, as compared with LPS alone, significant promoted TNF-α and IL-6 secretions, increased intracellular levels of TNF-α, IL-6 and iNOS (P<0.05), and reduced intracellular FKN, Wnt-4 and β-catenin levels (P<0.01). In FKN-overexpressing RAW264.7 cells, LPS treatment significantly reduced the secretion of TNF-α and IL-6 and intracellular levels of TNF-α, IL-6 and iNOS (P<0.01), increased intracellular FKN, Wnt-4 and β-catenin protein contents (P<0.01), and inhibited IL-6 localization in the cytoplasm; compared with LPS, the combined treatment with ICG-001 and LPS obviously enhanced IL-6 localization in the cytoplasm of the cells. Conclusion FKN overexpression suppresses LPS-induced M1 type polarization of RAW264.7 cells by activating Wnt/β-catenin signaling
pathway.

Key words: macrophages; polarization; fractalkine; Wnt/β-catenin signaling pathway